PET/MRI phantom studies are challenged by the need of phantom-specific attenuation templates to account for attenuation properties of the phantom material. We present a PET/MRI phantom built from MRI-visible material for which attenuation correction (AC) can be performed using the standard MRI-based AC.A water-fillable phantom was 3D-printed with a commercially available MRI-visible polymer. The phantom had a cylindrical shape and the fillable compartment consisted of a homogeneous region and a region containing solid rods of different diameters. The phantom was filled with a solution of water and [18F]FDG. A 30 min PET/MRI acquisition including the standard Dixon-based MR-AC method was performed. In addition, a CT scan of the phantom was acquired on a PET/CT system.From the Dixon in-phase, opposed-phase and fat images, a phantom-specific AC map (Phantom MR-AC) was produced by separating the phantom material from the water compartment using a thresholding-based method and assigning fixed attenuation coefficients to the individual compartments. The PET data was reconstructed using the Phantom MR-AC, the original Dixon MR-AC, and an MR-AC just containing the water compartment  to estimate the error of ignoring the phantom walls. CT-based AC was employed as the reference standard. Average %-differences in measured activity between the CT corrected PET and the PET corrected with the other AC methods were calculated.The phantom housing and the liquid compartment were both visible and distinguishable from each other in the Dixon images and allowed the segmentation of a phantom-specific MR-based AC. Compared to the CT-AC PET, average differences in measured activity in the whole water compartment in the phantom of −0.3%, 9.4%, and −24.1% were found for Dixon phantom MR-AC, MR-AC, and NoWall-AC based PET, respectively. Average differences near the phantom wall in the homogeneous region were −0.3%, 6.6%, and −34.3%, respectively. Around the rods, activity differed from the CT-AC PET by 0.7%, 8.9%, and −45.5%, respectively.The presented phantom material is visible using standard MR sequences, and thus, supports the use of standard, phantom-independent MR measurements for MR-AC in PET/MRI phantom studies. In the last decade, combined positron emission tomography/magnetic resonance imaging (PET/MRI) has been established in research and clinical practice . For thiMRI is based on the measurement of electromagnetic signals arising from the precession of the bulk magnetization of nuclear magnetic moments in an outer magnetic field after excitation by a radiofrequency pulse. In contrast to CT, the measured signal is not related to the attenuation properties of the investigated material for X-ray and annihilation radiation. Therefore, simple scaling approaches for AC, as used in PET/CT, cannot be applied and new AC concepts had to be developed. Today, several MRI-based AC methods are available, which work sufficiently well for assessing the attenuation properties of a human subject in most standard clinical PET/MRI investigations –9, althoMost solid materials, including those used for MRI coils, patient positioning devices or quality control phantoms, are not detectable by standard MRI , 15. TheTo overcome this issue, several techniques have been investigated. The most commonly employed one is the use of CT- or transmission measurement-based attenuation templates . Here, aFor flexible components and components for which the exact position is not known, template-based approaches including MRI-visible markers on the hardware components and non-rigid co-registration have been proposed . FurtherIn view of the challenges associated with the current approaches for AC of non-stationary hardware parts, and specifically phantoms, alternative approaches are desirable. One option is to visualize the hardware or phantom material with MRI and use simple segmentation-based methods for deriving the correct attenuation map. This would either require the use of ultra-short echo time (UTE) or zero echo time (ZTE) sequences, which are able to visualize solid materials with short relaxation times , 26. AnoThe phantom consists of a cylinder with an outer length of 19 cm, an outer diameter of 25.2 cm and a wall thickness of the cylinder shell of 6 mm. The cylinder was closed on the top and bottom with a 10 mm and a 5 mm lid, respectively. This results in a cylindrical inner compartment of 24 cm diameter and 18.5 cm length, which can be filled with liquids . The top lid contained a sealable drilling to allow filling the phantom. At the bottom lid, arrangements of rods of 5 cm length and with five different diameters of 5 mm, 10 mm, 13 mm, 17 mm, 22 mm, and 28 mm were attached. The chosen arrangement was similar to an MRI resolution phantom , but witwww.freecadweb.org). The design consisting of three parts, the cylinder, the top lid, and the test array was exported as an STL-file as an input for the 3D printer. For 3D printing, we used an MJM-printer Connex3 Objet 500 , which provides a resolution of 600 dpi for the x/y-coordinates and 800 dpi for the z-coordinate. The phantom was printed from high-temperature UV-curing material RGD525 in its pure quality. The printing of the model was done orthogonal to the axis of the cylinder with a layer thickness of 30 μm. Based on the dimension of the phantom, printing time was 79 h whereby 1.9 kg of the building material was needed.The whole phantom was constructed using an open source computer-aided design (CAD) program . The phantom was filled with a solution of water and 18F]fluoro-2-deoxy-d-Glucose ([18F]FDG) with an activity concentration of 10 kBq/ml. Sodium chloride (0.9%) and 5 ml MR contrast agent  were added to avoid MR artifacts . The pha8Ffluoro-2 using a 512×512 matrix.After the PET/MRI acquisition, a CT scan of the phantom was acquired on a Biograph TPTV PET/CT system . CT scan parameters were tube voltage = 120 kV, tube current time product = 160 mAs, pitch = 0.8, slice thickness = 5 mm, in-plane pixel size 1.4×1.4 mmFour different attenuation maps were created, whereby the standard, CT-AC map served as the reference standard. The standard Dixon-based MR-AC (“Standard MR-AC”) was generated using the method as implemented for whole-body AC in the PET/MRI system. The same data that were the base of the standard MR-AC approach were used to create a phantom-specific MR-AC (“phantom MR-AC”). Finally, an AC map only containing the water compartment  was created to imitate a typical phantom (as provided by the vendors), with a wall material that is not visible in standard MR-AC.Both, the phantom- and the NoWall MR-AC were created using a simple thresholding approach as follows. First, the total volume of the phantom was estimated from the “opposed-phase” MRI. A mask was produced by thresholding all pixels with values > 100 in the opposed phase MRI followed by a morphological closing operation to account for pixels with values < 100 within the phantom. The final mask representing the total phantom was defined by selecting the biggest connected compartment within the threshold mask followed by isotropic 1-voxel erosion. These steps were necessary to exclude artificial pixels at the edge of the MRI FOV and to account for an overestimation of the phantom extent caused by the low threshold, respectively.−1 and 0.1037 cm−1 to the water and phantom material, respectively. These values were obtained from the median of the linear attenuation coefficients from standard CT-AC. The NoWall MR-AC was created similarly with the only difference being that the phantom material was set to zero attenuation.Second, the water compartment was determined. The Dixon fat images were filtered with a Gaussian (sigma = 0.5 pixel) and all values < 10 where set to zero. Then the fat image was subtracted four times from the opposed phase image to enhance the contrast between the water and the phantom material compartment. This image was then used to produce a mask of the water compartment by thresholding all pixel values > 400 followed by a morphological closing operation to account for individual pixels with values < 400 within the water compartment. The phantom material compartment was obtained by subtracting the mask of the water compartment from the mask of the total phantom. The final phantom MR-AC was created by assigning a linear attenuation coefficient for 511 keV photons of 0.096 cmThe CT-AC was created by registering the CT images to the in-phase MRI and converting the CT units into linear attenuation coefficients using a bilinear scaling approach .PET data reconstruction was performed with the four AC maps. Reconstructions were done using vendor-based software  with an ordinary-Poisson ordered-subsets expectation maximization (op-OSEM) algorithm with 3 iterations and 24 subsets. Image matrix size was 344 × 344 × 127, and a 5-mm FWHM Gaussian post-reconstruction filtering was applied.AC maps and PET images were visually compared including an evaluation if the rod structure can be used to visually assess the PET resolution with the different AC methods similar as done in MRI or for single photon computed tomography (SPECT) using the Jaszczak Phantom .Voxel-based %-difference images between the different MR-AC maps and the CT-AC were calculated. Similarly, relative %-difference images between the MR-AC-corrected PET images and the CT-AC PET were calculated.To assess the impact of the different AC methods on quantitative PET readings, the average difference in activity concentration was calculated in the following regions of the phantom: (a) in the whole water compartment (by thresholding the CT-AC), (b) in a slice of the homogeneous region from an ROI including the whole water compartment in that slice, and additionally, in a 5 pixel wide ROI at the border of the phantom walls. (c) In a slice of the heterogeneous region from an ROI including the whole water compartment in that slice, and additionally, in a 5 pixel wide ROI around all rods and the phantom housing.The phantom housing and the liquid compartment were both visible and visually distinguishable from each other in the Dixon in-phase and opposed-phase images Fig. . However−1 was assigned. In general, the standard MR-AC overestimated the phantom extent in the order of one pixel (2 mm).The standard MR-AC included the phantom material, but did not separate the liquid compartment from the phantom material. The entire phantom was treated as soft tissue and a corresponding linear attenuation coefficient of 0.1 cmThe segmentation approach used for the phantom MR-AC did separate the phantom material from the water compartment. However, an overestimation of the extent of the phantom material within the phantom of in general about one pixel was seen Fig. . This ovPET images reconstructed with the standard and phantom MR-AC were visually comparable to the CT-based reconstructions. The rod structures were visible and distinguishable from each other down to a rod diameter of 10 mm in the PET images except the ones corrected using the NoWall MR-AC Fig. . The NoWCompared to the CT-AC PET, average differences (±SD) in measured activity in the entire water compartment of −0.3 (±2.1) %, 9.4 (±1.9) %, and −24.1 (±12.9) % were found for phantom MR-AC, standard MR-AC, and NoWall MR-AC-based PET, respectively. The average difference of the measured activity in a slice of the homogeneous region was −0.5 (±2.3) %, 9.1 (±2.0) %, −23.1 (±11.7) %, respectively. The average difference of the measured activity in a slice of the heterogeneous region was 0.5 (±2.2) %, 9.1 (±1.7) %, and −37.6 (±21.4) %, respectively.Average differences near the phantom wall in the homogeneous region were −0.3 (±4.3) %, 6.6 (±1.9) %, and −34.3 (±16.8) %, respectively. Around the rods, activity differed from the CT-AC PET by 0.7 (±2.6) %, 8.9 (±2.2) %, and −45.5 (±22.4) %, respectively. The artificial conjunction of the rods in vertical direction in the phantom MR-AC did only minimally influence the PET quantification between the rods  were challenged by reproducing the exact geometry of the phantom as seen on CT images. The standard MR-AC overestimated the extent of the phantom by approximately one pixel (2 mm). The phantom MR-AC did overestimate the extent of the 3D-printed phantom material within the object which was more pronounced in direction perpendicular to the original MRI slices Figs.  and 5. TThe local inaccuracies in reproducing the phantom housing material extent are also assumed to cause the local over- and under-estimations near the phantom walls in the phantom AC-based PET Fig. . A refinThe main limitation of the presented approach is that the phantom MR-AC segmentation procedure has been developed and tested only for one phantom. Specifically, the selection of the used thresholds was based on an empirical inspection of the voxel value distributions. To generalize the segmentation procedure for the use with a broad range of phantoms, the segmentation and threshold selection would require an optimization using a variety of phantoms with different shapes, wall thicknesses, and sizes. Furthermore, the applied morphological closing algorithm may cause artifacts when using phantoms containing air-filled cavities. However, with an optimized MR-AC sequence with improved homogeneity and improved separation of the water and phantom housing compartment, the morphological closing may not be needed.The presented MRI-visible polymer phantom housing is detectable using standard Dixon-based MR-AC sequences, and thus, can be incorporated in a routine MR-AC algorithms. When combining standard Dixon-based imaging with a dedicated thresholding approach, phantom-specific MR-AC maps were created that yielded a residual bias of the AC-PET of 1% or less.


The Occlutech Figulla occluder has been proven safe and effective at midterm follow-up after percutaneous atrial septal defect (ASD) closure. We describe the safety and efficacy at long-term follow-up in adults.  All consecutive adult patients that underwent ASD closure between 2008 and 2015 were included. All complications were registered. Residual left-to-right shunt (LRS) was diagnosed using color-Doppler transthoracic echocardiography (TTE). Right-to-left shunting was diagnosed using contrast TTE. Successful closure was defined as no LRS at follow-up.  In total, 166 patients  underwent percutaneous ASD closure using the Occlutech Flex I (70%) or Flex II (30%) device  under general anaesthesia and transoesophageal echocardiographic guidance. Long-term follow-up data were available for 144 patients (87%) with a mean follow-up of 5.9 ± 2.6 years, a total of 814 patient-years. During hospitalization, device embolization occurred in three patients (1.8%) with successful extraction in all. During the long-term follow-up, 15 patients (9.8%) suffered new-onset atrial fibrillation and stroke occurred in 2.1%. There was no residual LRS at 12-month follow-up. No device embolization occurred during the long-term follow-up.  Percutaneous ASD closure using the Occlutech device appears to be safe at long-term follow-up with a high successful closure rate at one year. Atrial septal defects (ASDs) are a common cardiac congenital finding with complications due to the left-to-right shunt (LRS). Percutaneous closure of an ASD is considered to be the first choice and has been proven safe and effective using different devices , 2. WorlAll consecutive adult patients, ≥18 years of age, that underwent percutaneous ASD closure with the Occlutech Figulla ASD Flex I or Flex II device (Occlutech®) in St. Antonius Hospital, Nieuwegein, Netherlands, between 2008 and 2015 were included. The local ethics committee approved the study (registration number R&D/Z16.054).Percutaneous closure was performed under general anaesthesia using transesophageal echocardiography (TEE) and balloon sizing as reported earlier . DependiAll complications were registered. All patients were discharged on antiplatelet therapy: clopidogrel 75 mg) for 4 weeks and aspirin (80–100 mg) for at least 6 months. If a patient was under anticoagulation therapy, only clopidogrel was associated for 4 weeks. Routine follow-up after closure was scheduled at 1, 6, 12, and 24 months using color-Doppler echocardiography to determine the presence of a residual LRS. Bubble contrast, at rest and after Valsalva, was used to identify a right-to-left shunt (RLS). The size of the RLS was classified by the number of bubbles in the left ventricle on a still frame and graded as minimal (<30 bubbles), moderate (30–100 bubbles), and large (>100 bubbles) [5 mg for Descriptive statistics were used for patients' characteristics. Continuous variables with normal distribution are presented as mean ± standard deviation. All statistical analyses were performed using the SPSS software (version 24.0 for Windows).In total, 166 adult patients  underwent percutaneous ASD closure using the Occlutech Figulla device between 2008 and 2015. During that study period, no other closure device had been used. The Occlutech Figulla Flex I was implanted in 116 patients (69.9%) and the Occlutech Figulla Flex II in 50 patients (30.1%). There were no differences in baseline characteristics between patients who received the different devices. Patient characteristics of the whole group are summarized in Device implantation  was initially successful in all patients. However, device embolization occurred in three patients (1.8%) within 24 hours after implantation. After a failed attempt to retrieve the devices percutaneously, they were extracted surgically followed by closure of the ASD. Balloon sizing ) was performed in almost all of the patients. However, another sizing balloon ) had been used, in 2 out of the 3 device embolizations, leading to an inappropriate sizing due to inadequate calibration. In the other patient, a deficient aortic rim was present.One patient (0.6%) suffered a significant amount of pericardial effusion (PE) a few hours after closure and needed percutaneous drainage. The next day, a second TTE showed no PE. The drain was retrieved, and the TTE was repeated a couple of hours thereafter. Again the TTE showed no PE. The patient was discharged, and after one and four weeks, no recurrent PE was found by TTE.At 6-month follow-up, device thrombosis occurred in one patient (0.6%) who was noncompliant for oral anticoagulation therapy prescribed for recurrent idiopathic deep venous thrombosis. The device was extracted surgically, followed by closure of the ASD. All patients in whom a complication occurred recovered well. There were no significant differences in complications between both devices. In-hospital complications are shown in In total, new-onset AF occurred in 9.8% of the patients during the overall follow-up. Recurrent thrombo-embolic events occurred in seven patients  during the total follow-up of almost 6 years. Details of these patients are summarized in Six patients died during follow-up. Three patients died in 15 months, two in 18 months, and one in 33 months after closure, respectively. One patient died due to the complications of liver cirrhosis. This patient had no evidence of pulmonary hypertension on TTE prior to closure. Another patient died because of systemic inflammatory response syndrome due to orbital cellulitis. He had a slightly elevated right ventricular systolic pressure (RVSP 40 mmHg) prior to closure. The third patient died after a tuberculosis infection. Before closure, this patient had an elevated RVSP (46 mmHg), which decreased after closure to 34 mmHg. A 70-year-old male, with a history of arterial hypertension and coronary artery disease, died due to sever systolic heart failure after myocardial infarction. There was no thrombus on the device and no elevated RVSP or secondary signs of pulmonary hypertension prior to closure. The cause of dead of the other two patients is unknown. No device-related cause was suspected after interviewing the families and general practitioners. One of these patients had a RVSP of 43 mmHg without secondary signs of pulmonary hypertension, and the other patient had normal pressures and no signs of pulmonary hypertension either. However, no autopsy was performed. Therefore, a device-related cause could not be excluded.TTE was performed in 71 patients (49.3%) at 12-month follow-up. Follow-up information was available for 144 patients at long-term follow-up (mean 5.9 ± 2.6 years); data (interview or TTE) could not be retrieved for 22 patients. There was no recurrent LRS at the latest follow-up. There were no significant differences between both devices at long-term follow-up. Long-term follow-up data are presented in Percutaneous ASD closure using the Occlutech device is safe and effective during a long-term follow-up of more than 800 patient-years.In the current literature, an overall initial successful device implantation using the Occlutech device had been described between 94% and 99% of the procedures –4, 8, 9.In our study, implantation was successful in 98%. Embolization occurred in 1.8%, which is similar when compared to the other studies. All three devices were extracted surgically, and the ASD had been closed without complications. There was one patient, noncompliant for Coumadin, who suffered a device thrombosis (0.6%). One patient suffered pericardial effusion without evidence of device perforation. As mentioned above, there were no significant differences in complications between the second- and third-generation devices. As mentioned earlier, Haas et al. described a large patient group of both children and adults. The median age was 26 years (range 0.3 to 83 years), which is significantly lower than our population. Furthermore, the first-generation Occlutech septal occluder (OSO) was used in 42% of patients. In our study, only second- and third-generation devices were used. Though, our results are similar to those described by Haas et al., cautiousness is necessary when comparing both studies.In the literature, cerebrovascular events after closure are rare and occur between 0% and 2.3% , 8–11. NNew-onset AF or atrial flutter is a known complication after percutaneous ASD closure and varies in studies between 0% and 4.7%. Supraventricular tachycardias (SVT) occurred in 4.7% in the study by Haas et al. . There wIn our study, new-onset AF occurred in 5.4% during the first year and in 4.4% at long-term follow-up, which is slightly higher when compared to the literature. A possible explanation is the higher age of our patients when compared to the other studies. There were new-onset SVTs in 11.7% in the study by Wang et al., which is much higher than the other studies . There wSuccessful closure using the Occlutech device varies between 90% and 100% at different follow-up times , 3, 8, 9Our study was an observational, single-center study describing the Occlutech device without comparison to other devices. Further, TTE was used at follow-up, making it possible to underestimate the residual LRS rate. However, it is unclear whether such a small LRS would be of clinical importance. There are no data about residual LRS at long-term follow-up, but TTE at discharge or one-year follow-up showed no LRS, making it unlikely that it would be found at long-term follow-up. We used telephone interviews for obtaining long-term data. This might be insufficient, and data about TTE are therefore not available.Percutaneous ASD closure using the Occlutech device has a high successful closure rate at 12 months and appears to be safe at long-term follow-up with a low complication rate. However, more follow-up data are needed to make a reliable conclusion.


However, the calibration of low-cost sensor measurements in an environment with high relative humidity is critical. This study proposes an efficient calibration and mapping approach based on real-time spatial model. The study carried out spatial calibration, which automatically collected measurements of low-cost sensors and the regulatory stations, and investigated the spatial varying pattern of the calibrated low-cost sensor data. The low-cost PM2.5 sensors are spatially calibrated based on reference-grade measurements at regulatory stations. Results showed that the proposed spatial regression approach can explain the variability of the biases from the low-cost sensors with an R-square value of 0.94. The spatial calibration and mapping algorithm can improve the bias and decrease to 39% of the RMSE when compared to the nonspatial calibration model. This spatial calibration and real-time mapping approach provide a useful way for local communities and governmental agencies to adjust the consistency of the sensor network for improved air quality monitoring and assessment.The data quality of low-cost sensors has received considerable attention and has also led to PM However, the traditional existing air quality monitoring networks are sparsely deployed and lack the measurement density, because the installation and maintenance of air quality monitoring instruments are expensive. In the race to develop high-resolution, spatiotemporal air pollutant monitoring systems, low-cost air quality sensors are promising supplements to regulatory monitors for PM2.5 exposure assessment2. Low-cost sensors have been used to collect real-time high-density air pollution data4. Low-cost sensors for PM2.5 were developed from Internet of Things (IoT)5. Investigators can also deploy additional sensors to increase spatial coverage of air quality monitoring network6. Moreover, low-cost sensors can gather air quality information of the community within real time at any location. The sensors are potentially easy to use and maintain because they require less energy and space to operate7.Air pollution is a severe global problem, especially in developing countries. A major air pollutant in urban environments is fine particulate matter  calibrated PM2.5 from a reference instrument with Federal Equivalent Method (FEM) status. However, keeping the regional sensors well-calibrated during deployment is a challenge9. Few approaches have been used to create field calibration formulas15. For example, linear models are used to adjust the raw data in certain cases16. Furthermore, nonlinear equations or machine learning methods are necessary to improve lower-cost sensor performance for air quality monitoring18. The reduction of measurement errors of large-scale low-cost sensor data is difficult with current multivariate calibration models2. To avoid regional misunderstanding of the measurements of low-cost air quality sensors, the development of a spatial calibration procedure is necessary. Thus, in the current study, we propose a real-time, regional, and simple way for low-cost sensor calibration based on reference-grade measurements at regulatory stations.To minimize low-cost sensor bias and uncertainties, calibration is essential for producing validated data. Calibration should be processed before, during, and after the data collection2.5 concentration map from calibrated low-cost air quality sensors. The real-time calibration function would vary with the specific location. As a basis for the spatial calibration model, the function was assumed to be related to locations, and the coefficient estimation was carried out in spatial regression. Moreover, regional PM2.5 concentration estimations were conducted after the calibration. Hence, the spatial calibrated concentration from low-cost sensors would be able to identify the reliable regional pollution hotspots.The objectives of the study are to develop the spatial varying relationship between the low-cost sensors and regulatory stations for calibration; to calibrate regional, low-cost air quality sensors against regulatory stations at one time slice; and to estimate the reliable PM2.5 sensor was launched in 2015, cooperation between the Academia Sinica and businesses, e.g., Edimax, led to the installation of thousands of boxes in Taiwan wherein local communities have volunteered to install low-cost PM2.5 sensors. The majority of air quality detection sensors, called AirBox, used the Realtek Ameba development board with the PMS5003 optical particulate matter sensor5. In this case, 2963 low-cost sensors were set-up , and relative humidity (%), were updated roughly every 5 min.As the program for the low-cost PM2.5  has been regularly recording the air quality and meteorological data throughout Taiwan. Since August 2005, the TWEPA has completed the installation of 76 automatic monitoring instruments for PM2.5 Fig. . The cur2.5 hourly data obtained by the TWEPA’s Air Monitoring Network and low-cost sensor real-time data were obtained for the fine aerosol concentration in Taiwan. The time-slice at 12:00 pm (UTC), 2020-02-24 is used as a case study. At that time, the relative humidity was high, and the air quality was poor from high PM2.5 concentrations in the western part of Taiwan due to weak diffusion condition. The average relative humidity is 82.1% and average temperature is 22 °C in the low-cost sensors. In high relative humidity, problematic observations from the low-cost sensor are expected to be found. Temperatures between 17 and 27 °C were mostly insignificant factors of sensor bias in the study area7.In this case study, the PM2.5 , and 2.5 concentration of regulatory sensors from TWEPA. The low-cost sensor calibration from nonspatial calibration model  is expressed as.The spatial regression coefficients, i is derived from weighted least-squaresi and j. The weight element can be computed as a kernel functioni is defined asi, and i. To evaluate the model performance, the common measures, R2 and RMSEs, are used based on the observation data and the estimated values at the regulatory stations.In the spatial adjustment, the estimated spatial coefficient 2.5 concentration map. The IDW method is used in this study to generate a 2 km-resolution PM2.5 concentration map after spatial calibration. The weights of the observations in the IDW are based on the inverse distances of the unknown point and observations. The parameter power value is 2 as the inverse distance squared weighted interpolation.Furthermore, the inverse distance weighting (IDW) method is a straightforward and low-computational approach for spatial interpolation procedures of the PM2.5 concentrations from (a) low-cost sensor and (b) regulatory stations and their corresponding histograms (c and d) are shown in Fig. 3, but the peaks of raw regulatory station data are 10–20, 40–50 μg/m3, respectively. In addition, the average values of low-cost sensors and regulatory sensors are 51.9 and 36.7 μg/m3, and the standard derivations are 26.1 and 17.5 μg/m3, respectively. The average values of the low-cost sensor data are double than those of the regulatory sensors. Furthermore, the variance of low-cost sensor data is significantly larger than the EPA monitoring data (681 and 306 (μg/m3)2 in the low-cost sensor and EPA data, respectively). The low-cost sensor data calibration is needed in the condition. Most PM2.5 measurements from raw low-cost sensors and regulatory stations showed extremely high correlation with the time series, and these can be used to identify the PM2.5 pollution hotspots7. However, the correlation of low-cost sensors and regulatory sensor data is only 0.8 from the collocated stations in this time slice.The raw PM3 after calibration from the nonspatial model, but can be improved to 4.1 μg/m3 using the spatial calibration model. Considering the spatial calibration model, the model performance improved, that is, the spatial scheme can reduce the inconsistency of low-cost sensors and the neighboring regulatory observations. Figure 3, respectively, and the standard derivation decreased from 26.1 to 18.0 μg/m3. The distribution of calibrated data in the spatial model is transformed to similar data distribution of regulatory stations. The result shows that the spatial calibration model performed better than the nonspatial calibration model.The nonspatial and spatial calibration models have R-square values of 0.64 and 0.94, respectively , appropriate automated correction routines must be established to remove problematic observations from the datasets or transform these observations based on regulatory stations1. Increased relative humidity is associated with a near-exponentially increased low-cost sensor data bias13. The observed influences of high humidity on low-cost sensor bias may be related to the issues in electronic circuits and the hygroscopic growth of fine particulates2. In a time series, the PM2.5 bias and relative humidity show positive association, while the relative humidity is high, particularly over 85%7. In this case, 43% of sensors exist with the relative humidity over 85%, and the total of 56% sensors exist with the relative humidity over 80%. We easily faced this condition because the area had humid subtropical climate in the north and a tropical monsoon climate in the south of Taiwan. Therefore, low-cost sensors with high relative humidity must be established to eliminate the possibility of problematic observations. Multi-variable models may be fitted to obtain reasonable formulas, which can be easily interpreted and implemented from each sensor22. However, the current spatial calibration model performs the best (4.8 μg/m3), because the model is already considered a local weighting for the spatial heterogeneity of calibration. Not considering the environmental variables further may simplify the low-cost sensor data spatial calibration. When considering the relative humidity into the spatial calibration model, the model RMSE increases to 5.4 μg/m3. This result implies that the relative humidity shows a negligible association with the spatial calibration. The relative humidity is not the influencing factor in the spatial model, because the high relative humidity spreads everywhere at this time (average relative humidity is 82.1%).Previous studies highlighted the association of the environmental conditions with the observation biases in low-cost sensors, as well as the calibration function that must be changed across space and time23. In practice, an appropriate and simple choice is to convert the low-cost sensor data to the reference system. The flexibility of an empirical spatial (location-specific) adjustment resulted in the consistent performance. After effective calibration, distributed low-cost sensors can help understand the spatial details of air pollution. In addition, data calibration commonly requires the space–time homogeneity of environmental conditions; however, the suitability of homogeneous assumption could not easily be validated7. If the environment is with high relative humidity, the real-time calibration for low-cost sensors is necessary. The calibration requires the real-time referenced and low-cost sensor data. The proposed approach did not find the general calibration equation, but needed to calibrate spatially at any time. In practice, the low-cost sensor will be real-time adjusted. In this case, computational time loading of the model is low (< 5 min) in the computer with Intel Core i5-10210U. The associations among low-cost sensors can be automatically converted to the reference system. In this case, the frequency of the monitoring systems of low-cost and regulatory sensors maybe inconsistent. The low-cost sensor updated rate is up to 5 min but the regulatory station is hourly updated. Thus, the real- or near real-time operation can only be set as the hourly calibration, then the real-time mapping of PM2.5 can be applied based on the current data with hourly-calibrated functions.Meanwhile, the calibration limitations and the risk of overfitting exist in small calibration datasets3. However, several types of low-cost sensors are also available for air quality monitoring in which the calibration may be applied diversely. In this spatial calibration model, the location-based and individual calibration coefficients at any time are considered.Low-cost sensors are cheap, compact, user-friendly, and provide high-resolution spatiotemporal air pollutant concentration data2.5 measurements from raw low-cost sensors can induce the biases of the risk perception from the public7. Even though the bias can be significantly improved through spatial calibration, the residuals can still be present in the result. For example, Fig. 2.5 better when compared to using regulatory stations. The pollution hotspots which were measured from the calibrated low-cost sensors happened in the central and southwestern areas of Taiwan.This study offers an effective approach for exploring the low-cost sensor data transformation using the spatial calibration model, which is a local weighting method and is easy to fit between low-cost sensor and regulatory stations. The spatial calibration and mapping model provide additional details on the spatial variations of sensor transformation, which can be identified from the nonlinear and spatially varying relationship between the low-cost sensor and regulatory stations. Given that the observations of low-cost sensors play a critical role, the bias of the PMThe study conducted spatial calibrations, which collected measurements of and investigated the spatial varying relations of low-cost sensors and regulatory stations. This work also presented the derivation of the spatial calibration approach for low-cost sensors given that the observed low-cost and regulatory measurements exhibited inconsistency and spatial heterogeneity.2.5 concentrations decreased to 4.8 μg/m3. The spatial calibration model can reduce the bias and decrease the mapping of RMSE to 39% compared to nonspatial calibration. Moreover, the use of regional maps after spatial calibration—instead of regulatory stations—provided spatial information on air pollution. Spatial pattern of air pollution showed the reliable hotspots of air pollution in the central area and in the southwestern area of Taiwan. The proposed model provides planners with relevant information by which to understand air pollution after the spatial calibration of low-cost sensors based on regulatory observations. The spatial calibration and mapping model can effectively mitigate the misleading pollution risk perceptions from decreasing the biases of low-cost sensors in the environment with high relative humidity. In the future work, we will consider more spatial information, such as elevation into the kernel function. In addition, the model is available for both real-time calibration and offline calibration. The offline calibration will be applied based on long-term monitoring data.The proposed model considered spatial regression and IDW interpolation for calibrating and mapping regional air quality. The low-cost sensor data can be adjusted using spatial calibration, especially in the environment with high relative humidity. Results showed that the proposed approach can explain the variability of the biases from the low-cost sensors with an R-square value of 0.94. The mapping RMSE values of PMhttps://sites.google.com/gs.ncku.edu.tw/ttest/%E9%A6%96%E9%A0%81Real-time visualization is available at


Eryngium caeruleum, we have evaluated its antidiabetic and antioxidant potentials. Both in-vitro and in-vivo assays have been carried out for antidiabetic assays. The antioxidant activity was determined by using different free radicals -6-sulfonic acid (ABTS), and hydrogen peroxide (H2O2)]. Moreover, different phytoconstituents were identified in the most active solvent fraction by GC-MS analysis. Furthermore, comparative fingerprints of methanolic extract and chloroform fraction were also analyzed via High Performance Liquid Chromatography coupled with Diode Array Detector (HPLC-DAD). The crude methanolic extract of E. caeruleum (Ec.Cr) and its sub-fractions . In the α-glucosidase inhibition assay, a concentration-dependent inhibitory response was observed against the enzyme. The most active sample was Ec.Chf which revealed an IC50 of 437 μg/ml in comparison to the standard acarbose (IC50 25 μg/ml). The rest of the samples showed moderate inhibition of α-glucosidase. In antioxidant assays, Ec.Chf and Ec.Cr exhibited a considerable scavenging effect against all the free radicals. The IC50 values recorded for Ec.Chf were 112, 109, and 150 μg/ml against DPPH, ABTS, and H2O2 respectively. Based on the in-vitro potential of Ec.Chf, this was subjected to the in-vivo model experiment. The Ec.Chf lowered the blood glucose level up to 10.3 mmol/L at 500 μg/Kg. The Ec.Chf was also subjected to GC-MS analysis. The GC-MS analysis confirmed the presence of 60 compounds. The identified phytoconstituents consist of some essential compounds previously reported with antidiabetic and antioxidant studies, which include thymol, tocopherol, phytol, nerolidol, (I)-neophytadiene, linolenic acid, and falcarinol. Similarly, the HPLC-DAD chromatograms of Ec.Cr and Ec.Chf exhibited a variety of peaks, which further demonstrates the possibility of important phytochemicals. In a nutshell, we can conclude that Eryngium caeruleum is a potential source of bioactive compounds which may be beneficial for the management of ailments like diabetes and free radicals mediated disorders. Molecular docking was performed to explore the possible role of all the identified bioactive compounds in the chloroform fraction of Eryngium caeruleum into active sites of the homology model of α-glucosidase.Natural-based drugs are believed to be safe, effective and economical. Based on the medicinal importance of the genus Eryngium and unexplored nature of  Diabetes mellitus (DM) is a disorder of carbohydrate, fat and protein metabolism attributed to low production of insulin or mounting resistance to its action leading to persistent hyperglycemia. DM affects about 5% of the population globally and its management exclusive of any side effects is still an issue that confronts the scientific community  called free radicals, which subsequently cause lipid peroxidation, membrane damage and play a vital role in the initiation of secondary complications of kidneys, eyes, blood vessels, nervous system, and DM  2,2-diphenyl-1-picrylhydrazyl (1898-66-4), 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (30931-67-0), Hydrogen peroxide, glibenclamide (10238-21-8), Ascorbic acid (50-81-7).The solvents used in the study were of analytical grades which were purchased from Sigma Aldrich via local venders. The solvents used in HPLC analysis were of HPLC grades. The chemicals and drugs used were purchased from the local suppliers of Sigma Aldrich. The chemicals and drugs used were α-glucosidase (9001-42-7), alloxan monohydrate (ALX2244-11-3), Acarbose (56180-94-0), Eryngium caeruleum were collected from various areas of the Malakand division and authenticated by Dr. Nasrullah at Department of Botany, University of Malakand. The plant was pressed and kept in the herbarium of University of Malakand, Pakistan under voucher specimen number H.UOM.BG.109. After collection, the plant was cut into small pieces and allowed to dry in shade. After drying, the plant was powdered using a cutter mill and macerated in 80% methanol for two weeks. After soaking, the plant sample was filtered using Whatman filter paper. The filtrates were evaporated under reduced pressure using rotary evaporator at 40°C . The fractions obtained for n-hexane, chloroform, ethyl acetate and aqueous fractions of E. caerulum were allowed to get converted into semisolid masses weighing 62 g (20. 66%), 37 g (12.33%), 59 g (19.66%), and 110 g (36.66%) respectively.The successive solvent-solvent extraction method was followed for the fractionation of crude methanolic extract  was made in the same buffer (pH 6.9) which acted as substrate for the reaction. Test samples prepared were mixed with the enzyme solution and incubated for 15 min at 37°C. Finally, 20 μl substrate solution was added to the enzyme mixture and was incubated for 15 min at 37°C. The reaction was completed by the addition of 80 μl of 0.2 M sodium carbonate solution. Absorbances were measured at 405 nm using UV spectrophotometer . The system without α-glucosidase was used as the blank and acarbose was the positive control. Each experiment was conducted in triplicate and percent inhibitions were calculated using the following formula;The α-glucosidase inhibitory potentials of solvent extracts from DPPH free radical scavenging ability of test samples were tested according to previously reported procedures .Where A2S2O4) 2.45 mM were mixed and stored in a dark place at room temperature for about 12–16 h to obtain a dark colored solution. This solution was diluted using Phosphate buffer (0.01 M) pH 7.4 and absorbance value was adjusted to 0.70 at 734 nm. Finally, 300 μl solution of the test sample was added to 3.0 ml of ABTS solution in cuvette and was analyzed using a UV spectrophotometer  at 734 nm. Reduction in absorbances was determined following 1 min of mixing the solutions and analysis was continued for 6 min. Ascorbic acid was used as a positive control. The assay was repeated in triplicate and percentage inhibition was calculated using the formula;The ABTS free radical scavenging potential of Plant samples were tested following previously published procedure  scavenging capacity of plant samples were evaluated using the previously reported method  was added to the tubes and were properly mixed using vertex mixer. Absorbance of each sample was measured at 230 nm against the blank after 10 min. H2O2 scavenging activity was calculated using the equation;Hydrogen peroxide .In the The mice were divided into two groups . The samples were administered I.P in a concentration ranges of 250–2,000 mg/kg body weight. After the samples' administrations, the animals were critically observed for 3 days for any abnormal response or mortality.Initially, we administered alloxan monohydrate  intraperitoneally to the experimental animals for the induction of hyperglycemia. Meanwhile, a control group was also maintained by experimental animals which received an intraperitoneal dose of normal saline. After the alloxan monohydrate injection, the animals were sorted out into diabetic and non-diabetic animals by using a glucometer. The animals with high blood glucose level (diabetes) were subjected to further studies as per the previous reported procedure . The group I was the normal one given normal saline and having no diabetes. Group II was given Tween 80 solution and was the diabetic group. Group III was given a standard drug for the treatment of diabetes . Group IV was given different concentrations of the potent chloroform fraction  with HHP-5MS 5% phenylmethylsiloxane capillary column  equipped with an FID detector. The temperature of the oven was retained at 70°C for 1 min at first, and then raised at the rate of 6°C/min to 180°C for 5 min and to finish at the rate of 5°C/ min to 280°C for 20 min. Injector and detector temperatures were set at 220 and 290°C, correspondingly. Helium was utilized as carrier gas at a flow rate of 1 ml/min  with a HHP-5MS 5% phenylmethylsiloxane capillary column  prepared with an Agilent HP-5973 mass selective detector in the electron impact mode (Ionization energy: 70 eV) working under a similar experimental environment as illustrated for GC  were mixed with 1 gram of The determination of various compounds' peaks was carried out by means of an Agilent 1260 infinity High-performance liquid chromatography (HPLC) system, having basic parts like quaternary pump, auto-sampler, degasser and ultraviolet array detector (UVAD). The separation was achieved via Agilent Zorbax Eclipse XDB-C18 column. The gradients system comprises of solvent B  and solvent C . The efficient gradient program was started with 100% B at 0 min, 85% B at 5 min, 50% B at 20 min, 30% B at 25 min, and 100% C from 30 to 40 min  , which are summarized in The GC-MS analysis of the most active fraction . A total number of 60 compounds were identified in the GC-MS analysis of chloroform fraction . VariousE. caeruleum were analyzed via HPLC-DAD. The chromatograms obtained for methanolic extract and chloroform fraction has been represented in The methanolic extract and chloroform fraction of In the chromatogram of methanolic extract, various components are separated in different ratios. The ratios of some distinct peaks are summarized in via docking simulations  software package was used for this purpose. These putative bioactive compounds identified in the chloroform fraction of Eryngium caeruleum were tested in-vitro against α-glucosidase from Baker's yeast. The 3-D structure of Saccharomyces cerevisiae (Baker's yeast) has not yet been reported. Hence, we performed docking simulations by using our previously reported Homology modeled α-glucosidase . One of the reasons for their effectiveness might be the solubility of active polar organic compounds in it. The remaining aqueous fraction was the least active. A major reason was that the majority of the active polar compounds tend to be extracted with chloroform and ethyl acetate, leaving a very small ratio of remaining compounds in the last fraction. For the preliminary identification of active compounds GC followed by GC-MS analysis of the active fraction was performed. We observed that 15 compounds were in greater concentration. Apart from the GC-MS analysis, further fingerprinting of E. caeruleum has been performed with the help of HPLC-DAD. The GC-MS and HPLC-DAD reveal the importance of E. caeruleum in terms of natural compounds.In the current study, we screened various solvent extracts from Experimental evidence implies the contribution of free radicals in the inception of DM and more significantly in the progress of diabetic complications . Compounds were only found active against ALR1 and ALR2 and were not able to show α- β-glucosidase inhibition and antiglycation potential. Docking studies were performed only on aldose reductase targets. In the current study, we performed a docking experiment to justify the synergistic effect of all these isolated bioactive compounds. The data obtained from docking studies agrees with the α-glucosidase inhibition activity. The computed binding energy of standard drug acarbose is −9.2756 kcal/mol. Putative bioactive compounds showed binding energy values in the range of −7.7008 to −4.5388 kcal/mol. Among simulated constituents, tocopherol showed best binding energy . While the binding energy from major phytoconstituents identified by GC-MS analysis were found in the range of −6.6118 to −4.0372 kcal/mol. The highest affinity was shown by bis(2-ethylhexyl) phthalate O in . The binEryngium caeruleum in the terms of antioxidant and anti-diabetic potentials followed by its GC-MS and HPLC-DAD analyses revealed that this plant is an excellent source of bioactive compounds. Based on the literature survey of identified compounds and ethnopharmacology of Eryngium caeruleum, it may be concluded that this plant is rich in antioxidant and anti-diabetic compounds and may be an effective remedy against various diseases, especially free radicals mediated and diabetic mediated disorders. Binding energy data computed via docking studies revealed that all putative bioactive compounds identified in the chloroform fraction of Eryngium caeruleum can inhibit α-glucosidase synergistically to treat hyperglycemia.The sorting out of the most active solvent fraction of The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Ethical Committee, Department of Pharmacy, Faculty of Biological Sciences, University of Malakand, Pakistan as per the animals Bye-Laws 2008 (Scientific Procedure Issue-I).in-vitro assays. IK helped in in-vivo experiment. MZ performed the HPLC analysis and helped in antioxidant assay. UR performed the molecular docking studies. MFA, RU, ON, Z-UI, and WA helped in GC-MS analysis and enzyme assay. AS supervised the whole project and drafted the manuscript for publication. All the authors have read the manuscript and approved for submission.SA, FU, and MA helped in The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Coronavirus disease 2019 (COVID-19) is currently the causative agent for a global health emergency and is predominantly associated with respiratory symptoms. In this case, a patient presented to the emergency department with gastrointestinal symptomatology without associated respiratory findings and was subsequently diagnosed with COVID-19 based on incidental findings from an abdominal computed tomography (CT) study. Given the patient’s lack of respiratory symptoms, diagnosis and treatment were ultimately delayed. During this global health crisis, an improved understanding of the various presentations of COVID-19 is paramount in an effort to initiate immediate treatment and prevent further transmission. According to the World Health Organization (WHO), on January 21, 2020, a novel type of coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first isolated on January 7, 2020 [When comparing the WHO Situation Report-1 to Situation Report-73 on April 2, 2020, there were 896,450 confirmed cases of coronavirus disease-2019 (COVID-19), with 45,526 deaths reported worldwide due to the disease outbreak [This data demonstrates that within 24 hours, 24,103 new cases and 996 deaths were reported in the US [We present the case of a 63-year-old obese Hispanic female with a past medical history only significant for rheumatoid arthritis and Sjogren's syndrome who presented to the emergency department with non-bloody diarrhea, nausea, vomiting, and right-sided abdominal pain radiating to her back for a week. The patient also reported having mild headaches but denied any cough or upper respiratory tract infection symptoms. While in the emergency department, the patient stated that she was dizzy. She was subsequently placed in observation for monitoring, where she was observed to have a persistent low-grade fever, measuring 100.9°Fahrenheit (F). Computed tomography (CT) of the abdomen was obtained and revealed patchy, peripheral multifocal ground-glass opacities at the lung bases Figure -b.On further investigation, the patient noted that she recently visited her sister in New York and that her sister also had chills, fever, and “pneumonia." The patient’s fever began two days after returning home from New York and she was discharged home after being evaluated at a nearby hospital at the time. Due to the ground-glass opacities seen on the abdominal CT examination, a fever for seven days, and a known history of a sick contact nine days prior, the patient was admitted for possible COVID-19 infection. Her low-grade fever of 100.9°F persisted for 24 hours in the hospital. The patient was initially started on lopinavir/ritonavir, ceftriaxone, and azithromycin. The patient ultimately tested positive for COVID-19 via nasopharyngeal swab. Subsequent chest x-rays showed progression of the ground-glass infiltrates Figure -b, and tChina reported cases of coronavirus within Wuhan, Hubei, China to the WHO on December 31, 2019 . The cauPan et al. observed that 50.5% of patients infected with COVID-19 reported gastrointestinal (GI) symptoms, including lack of appetite (78.6% of cases), diarrhea (34% of cases), vomiting (3.9% of cases), and abdominal pain (1.9% of cases), along with the presence of respiratory symptoms [The possible mechanism of liver tissue injury in COVID-19 may occur via the up-regulation of angiotensin-converting enzyme 2 (ACE2) expression in liver tissue -11. ThisLastly, gut microbiota plays a key role in the physiology of immune response , 13-14. Overall, it is imperative for clinicians to screen patients with GI symptoms for COVID-19 in an effort to manage and contain the SARS-CoV-2 virus efficiently. Currently, medical practitioners are screening patients for COVID-19 based primarily on the presence of fever and respiratory symptoms. Our patient is among the few accounts within the United States for which GI manifestations were her only presenting symptom. It is paramount that additional symptomatology with respect to the GI tract is noted as well given the known predilection for viral shedding and fecal-oral transmission. During this global health emergency, the identification of at-risk individuals through an improved understanding of viral presentation can enable immediate recognition and treatment before further transmission occurs.


L. plantarum (PLL-LP), incubated in a solution of pH 2 for 2 h, exhibited a higher viability (6.86 ± 0.12 log CFU/mL of viable cells) than non-coated L. plantarum (non-coated LP), which exhibited only 2.7 ± 1.23 log CFU/mL of viable cells. In addition, a higher amount of L. plantarum was detected in the feces of mice orally administered PLL-LP (6.2 ± 0.4 log CFU/g of feces) than in the feces of the control groups. In addition to enhancing probiotic viability in pH 2 solution, the PLL coating showed no effect on the probiotic growth pattern and the viability of either freeze-dried L. plantarum or L. plantarum, stored at −20 °C and 4 °C, respectively. Overall, these results indicated that the PLL coating is a promising potential probiotic delivery system.Due to their low acid tolerance, a majority of probiotics face difficulties with regard to surviving in the gastric environment long enough to reach the intestinal surfaces where they colonize and provide health benefits. We prepared a probiotic delivery system that can enhance their viability in acidic conditions by developing a one-step poly-L-lysine (PLL) coating process. We determined whether the coating process was successful by measuring the zeta potential and observing it with confocal scanning microscopy. PLL-coated  Since 2002, probiotics have been defined by the Food and Agriculture Organization and World Health Organization as “live microorganisms (bacteria or yeast) which when administered in adequate amounts confer health benefits to the host” . Recent The oral administration of probiotics products has been widely used to relieve specific symptoms and conditions of the GIT  . ProbiotSeveral forms of technology have been developed to address these difficulties. Typically, microencapsulation with various biomaterials is used to protect probiotics from direct contact with the external gastric environment, leading to an improved probiotic survival rate . Despite+ ion. Although a number of positively charged natural and synthetic polymers are used in drug delivery systems, only a few can be used for probiotic delivery systems because of the fragile viability of probiotics during the manufacturing process. For example, chitosan, the most widely used cationic polymer in probiotic microencapsulation systems, has an antibacterial activity, and therefore cannot be directly coated onto the surface of the probiotics without experiencing a decrease in its cationic charge. In the present study, we coated the surface of the probiotic bacteria Lactobacillus plantarum with poly-L-lysine (PLL), which was first identified in the culture filtrate of an actinomycete, Streptomyces albulus [L. plantarum is often isolated from various pickle preparations, and is widely used as a dietary supplement [L. plantarum was characterized by zeta potential measurement and confocal scanning microscopy, and the effect of PLL on L. plantarum was investigated by analyzing the growth pattern, freeze-drying stability and storage stability of the probiotic. In addition, the in vitro viability loss of poly-L-lysine-coated L. plantarum (PLL-LP) against the acidic pH of the gastric environment, and in vivo viability, were investigated in imprinted control region (ICR) mice using non-coated L. plantarum (non-coated LP) as a control.To overcome these drawbacks, we hypothesized that coating the surface of the probiotics directly with a cationic polymer by using a one-step coating process could be a promising method for developing a probiotic delivery system that enhances probiotic viability in gastric acid by electrostatic repulsion with H albulus . PLL is  albulus ,13. It w albulus . Previou albulus ,16. To tpplement ,18,19. Ipplement ,20,21. ATM Lactobacilli MRS Broth (MRS) and agar were purchased from BD biosciences . Bromocresol purple, ciprofloxacin and LIVE/DEADTM BaclightTM Bacterial Viability Kit were purchased from Thermo Fisher Scientific Inc. . Flamma® 648 NHS ester was purchased from BioActs . All other materials and solvents used were of the highest analytical grade.Poly-L-lysine (PLL) (average molecular weight: 4700) was kindly provided by JNC corporation  as a gift. Sodium chloride and L-cysteine were purchased from Sigma-Aldrich . BactoL. plantarum (KCTC 3108) was obtained from the Korean Collection for Type Cultures. The stock culture of L. plantarum was stored at −80 °C in MRS broth with 15% glycerol and activated by spreading onto an MRS agar plate containing L-cysteine. Then, it was incubated at 37 °C for 48 h under anaerobic conditions. A primary starting culture was prepared by transferring one colony from the incubated MRS agar plate to fresh MRS broth with L-cysteine and incubating it for 12 h. The working culture was then prepared with a 1:50 ratio of starting culture to fresh MRS broth and incubated at 37 °C for 24 h in a shaking incubator. The final concentration of L. plantarum in the working culture was approximately 1 × 1010 log CFU/mL. Before starting the coating process, the working culture was centrifuged at 2500 g for 10 min at 4 °C. The supernatant was discarded, and the pellet was washed three times with normal saline.Pure culture of L. plantarum viability was quantified by using the plate counting method with MRS agar [TM EZ, Sparks, NE, USA) according to the manufacturer’s instructions. All samples were independently produced in triplicates, and viable cells were counted in terms of colony-forming units (CFU) on the MRS agar plates.In this study, MRS agar . In detaL. plantarum were re-suspended in a sterilized PLL solution in which the pH was adjusted to 6 based on the pKa value of PLL, to protonate the PLL amino group. As a probiotic is a living cell, the selection of solvent, pH, temperature and concentration were carefully determined. After optimizing the method, the coating was applied by gently rotating the cells for 15 min. After rotation, the probiotic cells were pelletized by centrifugation at 2500 g for 10 min at 4 °C, and excess PLL solution was removed by washing three times with 0.15 M NaCl solution. The same rotation time and washing steps were performed with uncoated cells in 0.15 M NaCl solution as a control.As shown in L. plantarum surface with the positively charged polyelectrolyte PLL was characterized by determining the zeta potential with a Malvern Zeta analyzer, Nano-ZS90 . The zeta potential was measured using double-distilled water as a dispersing medium. Each data point was obtained by taking the average of five independent measurements and their standard deviations.Successful coating of the negatively charged L. plantarum was observed using a ZEISS LSM 800 confocal microscope  equipped with 63× magnification. First, freshly prepared non-coated LP and PLL-LP were each interacted with an equal amount of Flamma® 648 NHS ester dye for 1 h in the dark. Flamma® 648 NHS ester dye interacts with primary amine groups and forms strong amide bonds. As PLL is a homo-polypeptide of a lysine residue, it is rich in primary amine groups; therefore, a red signal indicates the presence of PLL. After their interaction with the NHS ester dye, free dye was removed by centrifugation at 2500 g for 10 min at 4°C three times with double-distilled water. Then, the pelleted cells were stained with SYTO 9 dye according to the manufacturer’s protocol, to observe the shape of the probiotics. The suspended cells were fixed on a glass slide and observed with a confocal laser microscope using excitation/emission wavelengths of 648/663 nm and 480/500 nm, to observe the fluorescence of the NHS ester and that of the SYTO 9, respectively.The coating of PLL on the surface of the The growth pattern of PLL-LP was determined, in order to evaluate the effect of the PLL coating on the growth of the cells. Both non-coated LP and PLL-LP were cultured anaerobically in MRS broth by shaking at 220 rpm in a shaking incubator at 37 °C. The turbidity of the bacterial solution, which indicates the increase in the growth of bacteria, was determined by measuring the optical density (OD) at a wavelength of 600 nm . The turTM BaclightTM Bacterial Viability Kit according to the manufacturer’s protocol. The samples were then observed with a ZEISS LSM800 confocal laser microscope with 20× magnification and excitation/emission maximum wavelengths of 480/500 nm and 490/635 nm, to observe the SYTO 9’s and the propidium iodide’s (PI) fluorescence, respectively.In this study, the viability of non-coated LP and PLL-LP in an acidic solution was determined. Specifically, a low-pH solution (pH 2) was prepared by using a 0.2% sodium chloride solution with diluted hydrochloric acid to simulate the highly acidic human stomach conditions. Both non-coated LP and PLL-LP were incubated in prepared pH 2 solution for 2 h. To evaluate the exact number (in log CFU) of viable non-coated LP and PLL-LP cells, the incubated probiotics suspended in gastric pH for 1 and 2 h were serially diluted and 100 μL of each dilution was spread onto MRS agar plates. The agar plates were incubated at 37 °C in anaerobic conditions for 48 h, and then the number of colonies on the agar plates was counted to confirm bacterial viability. Each data point was obtained by taking the average of eight independent measurements and their standard deviations. Additionally, after 2 h of incubation in an acidic solution, the probiotic cells were stained with the LIVE/DEADL. plantarum, the viability of non-coated LP and PLL-LP without added cell protectant was evaluated during the storage and freeze-drying processes. To determine storage stability at 4 °C, equal amounts of freshly prepared and pelletized non-coated LP and PLL-LP were put in separate storage tubes and stored at 4 °C in a refrigerator for 4 weeks. We also determined the protective effect of PLL against the freeze-drying process. Pelletized probiotic cells were first kept at −80 °C for 7 h and then subsequently lyophilized in a freeze-drier overnight. The freeze-dried samples were kept in a −20 °C refrigerator to evaluate their stability. The viability of the storage samples (at 4 °C and −20 °C) was evaluated weekly for 4 weeks.To confer the desired health benefits, a sufficient number of probiotics must survive not only the gastric conditions but also the final manufacturing processes . Viable 8 log CFU/mL of cells in distilled water by oral gavage. After 24 h, mice were housed individually, and feces was collected within 10 min of defecation. Each feces sample was put in a sterilized conical tube, weighed, and serially diluted with sterilized normal saline solution for the enumeration of viable cells. To avoid contamination with external microorganisms, all processes were operated with a sterilized apparatus and solvent. To avoid detecting other bacteria in the mouse feces, a selective agar plate for L. plantarum [Animal experiments were performed in accordance with the regulations of Pusan National University Institutional Animal Care and Use Committee (PNU-IACUC) . Male ICR mice  were chosen as the animal model. Animals were provided food and water and kept at 25 °C ± 3 °C in a 12-h light/dark cycle. Animals were divided into 3 groups of 4 mice each: the control (distilled water), non-coated LP, and PLL-LP groups. Mice in the LP and PLL-LP groups were administered 2 × 10lantarum ,27 was uP-values less than 0.05 were considered statistically significant.Statistical analyses of all in vitro and in vivo data were performed using two-way ANOVA and one-way ANOVA followed by the Bonferroni test in GraphPad Prism 5.0 . L. plantarum. Various concentrations of PLL  were coated onto the probiotics, and the zeta potential was measured. The zeta potential of L. plantarum changed from negative to positive (mV) when the coating concentration of PLL reached 1 mg/mL. PLL coating concentrations higher than 1.5 mg/mL produced no significant change in the zeta potential. This may be due to the limited surface area of L. plantarum; the excess PLL would have been removed by the washing process. In this study, we chose to use 1.5 mg/mL of PLL for further experimentation, as this concentration produced the highest positive charge.Unlike previous LbL technical methods and materials used for probiotic encapsulation systems, we used only one biopolymer to form a single-layer coating for the probiotic cells. We used the well-known mechanism of electrostatic adsorption to coat polycationic PLL onto the negatively charged cell surface of L. plantarum membrane would be destroyed due to the positive charge. Most of the cationic polymers widely used in drug delivery systems, such as chitosan and polyethyleneimines, also present this challenge because of their antimicrobial activity. Although PLL has been shown to inhibit the growth of microbial agents, the minimum inhibitory concentration (MIC) depends on the strain of bacteria, the pH, and the incubation time intervals [L. plantarum was evaluated after it was coated with different concentrations of PLL. As shown in L. plantarum during the 15-min incubation period. The viability of L. plantarum did not show any significant changes after being coated with a 1.5-mg/mL PLL solution . According to this result, the process of coating with a 1.5-mg/mL PLL solution did not affect the viability of L. plantarum.As PLL is a cationic polymer, there was a concern that the ntervals . To deteL. plantarum was coated with a 1.5-mg/mL PLL solution, as shown in L. plantarum with PLL was determined by staining with two fluorescents dyes. A red signal, indicating the presence of PLL, was observed throughout the PLL-LP, whereas no red fluorescence was observed in the non-coated LP, as shown in After optimizing the concentration and incubation time, L. plantarum did not influence the growth pattern of the probiotic in the presence of MRS medium at 37 °C in a shaking incubator. As shown in In our study, the single-layer PLL coating on L. plantarum in a solution of pH 2. L. plantarum incubated in an acidic solution. After 1 and 2 h of incubation in the solution of pH 2, non-coated LP showed a significant reduction in viability . Conversely, the viability of PLL-LP was reduced by only 2 log CFU and 3 log CFU after 1 and 2 h of incubation in a solution of pH 2, respectively.Here, we examined the viability of non-coated and PLL-coated In addition, confocal microscopy images of non-coated LP and PLL-LP after incubation in pH 2 solution for 2 h are shown in L. plantarum after storage at 4 °C was not related to the presence of the PLL coating.As shown in L. plantarum at −20 °C was evaluated for 1 month. As shown in To verify the protective effect of the PLL coating on the probiotic cell membrane, PLL-LP was lyophilized. The survival rate of PLL-LP was determined before and after the freeze-drying procedure and compared to that of non-coated LP. According to the results, as shown in L. plantarum via oral gavage. The feces of mice that received PLL-LP by oral gavage contained more colonies of L. plantarum (about 6.12 ± 0.4 log CFU/g of feces). Conversely, non-coated LP-treated mouse feces exhibited colony sizes of only 3.79 ± 2.5 log CFU/g of feces, and as expected, there were no L. plantarum colonies detected in the feces of the control group. The viability of lactic acid bacteria varies greatly among different people, and even among samples collected from the same subject [To determine the actual survivability of PLL-LP, animal studies have been performed using ICR mice. The survival of PLL-LP in vivo study was performed via administering with the same amount of non-coated and PLL-coated  subject ,32. In a subject ,34. AccoL. plantarum in acidic conditions by conducting a simple and brief one-step coating process with poly-L-lysine, which produced a coating capable of maintaining the growth and proliferation ability of probiotic cells. In addition, the PLL coating did not negatively affect the viability of L. plantarum after it was freeze-dried and stored. It was also determined that the coating enhanced not only the in vitro viability of L. plantarum in a gastric acid solution, but also in vivo viability, with little variation. Our results indicated that coating with PLL may be a potentially effective tool for improving the viability of L. plantarum. Our study presented a promising approach to designing a probiotic delivery system in the food and pharmaceutical industries.In this study, we significantly enhanced the in vitro viability of


Gal-7 was also expressed by tumor-infiltrating macrophages. High Gal-8 expression in the cytoplasm was associated with a significantly improved overall survival (OS) (p = 0.032). Clinical outcome in patients showing both high Gal-7 and with low Gal-8 expression was very poor. Further understanding of the role of galectins in the regulation and interaction of tumor cells and macrophages is essential for finding new therapeutic targets.Galectins are commonly overexpressed in cancer cells and their expression pattern is often associated with the aggressiveness and metastatic phenotype of the tumor. This study investigates the prognostic influence of the expression of galectin-7  and galectin-8  in tumor cell cytoplasm, nucleus and on surrounding immune cells. Primary breast cancer tissue of 235 patients was analyzed for the expression of Gal-7 and Gal-8 and correlated with clinical and pathological data and the outcome. To identify immune cell subpopulations, immunofluorescence double staining was performed. Significant correlations of Gal-7 expression in the cytoplasm with HER2-status, PR status, patient age and grading, and of Gal-8 expression in the cytoplasm with HER2-status and patient age and of both galectins between each other were found. A high Gal-7 expression in the cytoplasm was a significant independent prognosticator for an impaired progression free survival (PFS) ( Breast cancer is one of the three most prevalent cancers worldwide and the most frequent malignant tumor in women . In 2018Based on gene profiling studies, breast cancer can be classified into different intrinsic subtypes . In dailThe discovery of galectins in 1994  led to iIn this study, the specific role of Gal-7 and Gal-8 in a bigger cohort of human primary non-metastatic breast cancer was evaluated. The analysis included the specific location of galectin expression in the nucleus or cytoplasm and the expression in tumor-surrounding immune cells.Expression of both galectins was observed in the cytoplasm as well as in the nucleus, being more pronounced for Gal-7. Gal-7 expression could not be evaluated in 19 sections . Seven cases showed no Gal-7 expression in the cytoplasm and 35 no staining in the nucleus. For Gal-8, 20 tissue sections could not be analyzed , and 15 and 63 samples revealed no staining in the cytoplasm and in the nucleus, respectively. The distribution of the immunoreactivity score (IRS) for the staining in the cytoplasm and in the nucleus is shown in Both Gal-7 and Gal-8 expression in the cytoplasm and in the nucleus did not correlate with the clinical parameters: tumor size and lymph node status. Gal-7 and Gal-8 expression in the cytoplasm correlated negatively with patient age  (p = 0.042). The Gal-8 expression did not differ concerning the histological subtype.The Gal-7 expression correlated negatively with the histopathological subtype. Kruskal–Wallis test and boxplots analysis showed that Gal-7 expression in the cytoplasm was significantly higher in no special type (NST) tumors compared to non-NST tumors  a. Similap = 0.003, p = 0.089). Exemplary immunohistochemical Gal-7 and Gal-8 staining in tumors with different gradings are shown in Regarding tumor grading, a positive correlation with the cytoplasmic Gal-7 expression was found   : HER2-po< 0.001)  nor in tGal-7 expression in the nucleus and cytoplasm correlated to each other as well as Gal-8 expression in the nucleus and cytoplasm . Gal-7 aMedian overall survival (OS), progression-free survival (PFS) and distant disease-free survival (DDFS) in the whole cohort was not reached (NR). The prognostic relevance of Gal-7 and -8 was analyzed concerning PFS, DDFS and OS and tumors were categorized in “high” and “low” expressing tumors using ROC-curve analysis.p = 0.017, median PFS in Gal-7 high: 9.7 years, median PFS in Gal-7 low: NR)    was associated with a worse outcome: tumors with high Gal-7 expression in the cytoplasm showed a significantly impaired PFS (low: NR) a and DDFoups NR) b. Conceroups NR) c.Multivariate COX regression analysis revealed Gal-7 expression as an independent prognostic factor for PFS  but not p = 0.036, median PFS in Gal-7 high 2.52 years, p = 0.014) and in the pN0 (but not in pN > 0) (p = 0.043) subgroups. A trend towards an impaired PFS could be observed in PR positive (but not PR negative) (p = 0.098) and in HER2 positive (but not in HER2 negative) tumors   (but not in G2-3) when nuclear Gal-7 expression was high.Gal-7 expression in the nucleus did not show prognostic relevance for PFS, DDFS or for the OS . Subgroup = 0.032, median OS in both subgroups NR)  was associated with an improved outcome: Tumors with a high Gal-8 expression in the cytoplasm showed a significantly improved OS compared to tumors with low Gal-8 expression in the cytoplasm (oups NR) a. No sigThe multivariate COX regression analysis could not confirm Gal-8 expression in the cytoplasm as an independent prognostic factor  for OS. p = 0.049), in HER2-negative (but not HER2-positive) . Concerning PFS and DDFS, no significant prognostic impact could be found for Gal-8 in the different subgroups.Subgroup analysis revealed that Gal-8 expression in the cytoplasm had prognostic relevance with a better outcome for the OS in NST (but not non-NST) tumors (p = 0.041) but not in ER negative patients and to tendentially (p = 0.081) better OS in pT2-4 (but not in pT1) tumors. Regarding the other subgroups , nuclear Gal-8 staining did not show significant prognostic influence, similar to the overall cohort. Similar to Gal-7 staining in the nucleus, Gal-8 staining in the nucleus did not show prognostic relevance for neither PFS, DDFS or OS . Howeverp = 0.201, median OS in all subgroups NR, p = 0.009, median DDFS in all subgroups NR, p = 0.067, median PFS in all subgroups NR, Survival analysis was also performed in subgroups with high or low Gal-7 expression in the cytoplasm in combination with low or high Gal-8 expression in the cytoplasm, respectively. A high Gal-7 combined with a low Gal-8 expression in the cytoplasm was associated with an impaired OS compared to all other patients , whereas no effect could be shown for Gal-8  . Our results suggest that combining Gal-7 and Gal-8 expression might further improve prognostic accuracy. Two different groups could be defined: a small group  with a very good outcome (6.8% of the patients), compared to a second group  with a worse outcome, consisting of 13.6% of the patients. This is especially interesting, as Gal-7 and Gal-8 expressions are strongly correlated. Similar effects have been shown for galectin ligands: in BC patients, high levels of- Gal-1 ligands and low levels of Gal-8 ligands have been observed, making their ratio a strong marker for BC . A similGalectin expression was also associated with tumor cell differentiation: Gal-7 expression was significantly higher in less differentiated cells (reflected by higher grading), whereas Gal-8 expression was highest in highly differentiated cells (reflected by lower grading). This is similar to other data where high Gal-4  expression in highly differentiated, and low Gal-1  expression in poorly differentiated, tumors have been shown . FurtherRegarding mechanisms of the regulation of galectin expression and distribution between nucleus and cytoplasm, it is important to consider that Gal-7 is also provided extracellularly: The extracellular Gal-7 controls the intracellular pool of Gal-7, firstly by an increase in the gene transcription, and secondly by a re-entry pathway into the cells . SimilarFirst attempts in targeting galectins in breast cancer have already been made: Grosset et al. demonstrated that targeting CRD-independent cytosolic Gal-7 in breast cancer cells, and therefore impairing p53-functions, might be a valuable strategy for the treatment of breast cancer . TargetiFormalin-fixed, paraffin-embedded (FFPE) primary breast cancer samples of 235 patients were examined in this study .We included in our study all patients that were diagnosed with primary non-metastatic breast cancer and underwent surgery at the Department of Gynaecology and Obstetrics, Ludwig-Maximilians-University Munich, Germany, from the period 1998 to 2000. Therefore, there was no pre-selection and a complete group of patients attending the clinic was analysed. Only women with benign tumors of the breast were excluded from the study.Clinical, pathological and follow-up data (up to ten years) were retrieved from patients’ charts and from the Munich Cancer Registry. Patient characteristics are displayed in Histopathological subtype (no specific type (NST) vs. non-NST), tumor grading (G1-3) according to the Elston and Ellis criteria (1993) ,38,39, aEndpoints regarding the survival data were defined as follows: OS = overall survival, period of time from the date of surgery until the date of death or date of last follow-up; PFS = progression free survival, period of time until local recurrence or metastasis were diagnosed and DDFS = distant disease free survival: period of time until metastasis was diagnosed.Paraffine-embedded breast cancer tissue samples were analyzed by immunohistochemistry. The samples were fixed in neutral buffered formalin and embedded in paraffin after surgery. For histopathological investigations, tissue sections (3 µm) were deparaffinized in Roticlear (Carl Roth GmbH + Co. KG) for 20 min and then the endogenous peroxidase was inactivated with 3% hydrogen peroxide  in methanol. The slides were rehydrated in a descending gradient of ethanol  and prepared for epitope retrieval in a pressure cooker for 5 min in sodium citrate buffer . After washing in distilled water and phosphate-buffered saline (PBS), all tissue slides were blocked using a blocking solution ; Zytomed Systems GmbH, Berlin, Germany) for 5 min at room temperature (RT) in order to block non-specific binding of the primary antibodies. Then, a specific procedure followed for each Galectin: the slides were incubated with Gal-7 primary antibody  at a final concentration of 2.5 µg/mL in PBS Dulbecco (Biochrom GmbH) for 16 h at 4 °C. Gal-8 primary antibody  was used for incubation at a final concentration of 3.3 µg/mL in PBS for 1 h at RT. Afterwards, the staining specimens were incubated in post block reagent (Reagent 2) and HRP-polymer (Reagent 3) containing secondary antibodies (anti-mouse/-rabbit) and peroxidase according to the manufacturer’s protocol. These antibodies are part of the provided Reagent 3, exact concentrations are not specified by the manufacturer. . All slides were washed in PBS after every incubation step. The slides were then stained with 3,3′-diaminobenzidine chromogen  for visualization and counterstained in Mayer acidic hematoxylin. After dehydrating in an ascending ethanol gradient and Roticlear they were cover slipped with Roti-Mount (Carl Roth GmbH + Co. KG). Appropriate tissue slides were used as positive controls . To obtain expression results, the semiquantitative immunoreactive score  for 20 min, the slides were rehydrated in a descending gradient of ethanol  and prepared for epitope retrieval in a pressure cooker for 5 min in sodium citrate buffer . After washing in distilled water and PBS, all tissue slides were blocked with Ultra V Block (Thermo scientific) for 15 min. The primary antibodies were diluted in Dako Antibody Diluent (Dako North America) incubated with the slides for 16 h at 4 degrees. Gal-7  was diluted at a final concentration of 2,5 µg/mL and CD68  at a concentration of 0.1 µg/mL. Next, the light in the room was dimmed and an incubation with the secondary antibodies for 30 min at RT followed. Secondary antibodies: Goat-Anti Rabbit IgG Cy3  diluted at a concentration of 3 µg/mL and Goat-Anti-Mouse-AlexaFluor488-IgG  at a concentration of 15 µg/mL. After the slices were dried in the dark, they were cover slipped with mounting medium for fluorescence with DAPI (Vectashield H-1200). The samples were then analyzed using a Zeiss AxioPhot microscope with an Axiocam MRm within one day.p-values lower than 0.05 were considered as statistically significant. Correlations between staining results and ordinal variables were tested with Spearman’s rank correlation coefficient. Group comparisons regarding the IRS of galectins between different clinical and pathological subgroups were tested with Kruskal–Wallis test and displayed as boxplot graphs. Survival times between different groups were compared by Kaplan–Meier analysis, and differences were tested for significance by Log-Rank (Mantel-Cox), Breslow- and Tarone-Ware-tests. Censored cases are cases for which the second event is not recorded . The Kaplan–Meier procedure is a method of estimating time-to-event models in the presence of censored cases. The Kaplan–Meier model is based on estimating conditional probabilities at each time point when an event occurs and taking the product limit of those probabilities to estimate the survival rate at each point in time. Cox-regression analysis was used to determine the independence of prognostic factors.Data analyses were performed with SPSS Statistics 25 . Concerning survival analysis dependent on Gal-7 and Gal-8 expression, patients were grouped into high and low expression. Cut-off points were selected considering the distribution pattern of IR-scores in the collective. Therefore, the receiver operator curve (ROC curve) was drawn using SPSS software, which is considered as one of the most reliable methods for cut-off point selection. In this context, the ROC curve is a plot representing sensitivity on the y-axis and (1-specificity) the x-axis. Consecutively Youden index, defined as the maximum (sensitivity + specificity−1), was used to find the optimal cut-off maximizing the sum of sensitivity and specificity . The breast cancer specimens were obtained in clinically indicated surgeries. When the current study was performed, all diagnostic procedures were completed, and the patients’ data were anonymized. The ethical principles adopted in the Declaration of Helsinki 1975 have been respected. As per the declaration of our ethics committee, no written informed consent of the participants or permission to publish is needed given the circumstances described above. Researchers were blinded from patient data during experimental and statistical analysis.In summary, our results suggest that Gal-7 might be an independent negative prognostic factor in breast cancer and therapeutic target, especially in HER2-positive breast cancer. Furthermore, Gal-8 was observed to be a positive predictor for overall survival and upregulation should be further investigated. The role of Gal-7 and Gal-8 should be validated in a BC collective treated with today’s standard of therapy—even if this might be outdated at the time point of the analysis. Additional studies are required to detect the signaling pathways in which both Gal-7 and Gal-8 are involved, as the combination of both markers showed strong prognostic impact. Gal-7 and Gal-8, as well as the whole group of galectins, seem to be interesting therapeutic and prognostic targets that might help to improve therapies and outcome for breast cancer patients in the future.